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Nickel Nanoparticles Induce the Synthesis of aTumor-Related Polypeptide in Human Epidermal

KeratinocytesJavier Jiménez-Lamana, Simon Godin, Gerard Aragonès, Cinta Bladé, Joanna

Szpunar, Ryszard Lobinski

To cite this version:Javier Jiménez-Lamana, Simon Godin, Gerard Aragonès, Cinta Bladé, Joanna Szpunar, et al..Nickel Nanoparticles Induce the Synthesis of a Tumor-Related Polypeptide in Human EpidermalKeratinocytes. Nanomaterials, MDPI, 2020, 10 (5), pp.992. 10.3390/nano10050992. hal-02734588

nanomaterials

Article

Nickel Nanoparticles Induce the Synthesis of aTumor-Related Polypeptide in HumanEpidermal Keratinocytes

Javier Jiménez-Lamana 1,* , Simon Godin 1, Gerard Aragonès 2 , Cinta Bladé 2,†,Joanna Szpunar 1 and Ryszard Łobinski 1

1 Universite de Pau et des Pays de l’Adour, E2S UPPA, CNRS, IPREM UMR, 5254 Pau, France;simon.godin@univ-pau.fr (S.G.); joanna.szpunar@univ-pau.fr (J.S.); Ryszard.Lobinski@univ-pau.fr (R.L.)

2 Department of Biochemistry and Biotechnology, Nutrigenomics Research Group, Universitat Rovira i Virgili,43007 Tarragona, Spain; gerard.aragones@urv.cat

* Correspondence: j.jimenez-lamana@univ-pau.fr; Tel.: +33-540175037† Deceased.

Received: 23 April 2020; Accepted: 19 May 2020; Published: 21 May 2020

Abstract: Although nickel allergy and carcinogenicity are well known, their molecular mechanismsare still uncertain, thus demanding studies at the molecular level. The nickel carcinogenicity is knownto be dependent on the chemical form of nickel, since only certain nickel compounds can enter thecell. This study investigates, for the first time, the cytotoxicity, cellular uptake, and molecular targetsof nickel nanoparticles (NiNPs) in human skin cells in comparison with other chemical forms ofnickel. The dose-response curve that was obtained for NiNPs in the cytotoxicity assays showed alinear behavior typical of genotoxic carcinogens. The exposure of keratinocytes to NiNPs leads tothe release of Ni2+ ions and its accumulation in the cytosol. A 6 kDa nickel-binding molecule wasfound to be synthesized by cells exposed to NiNPs at a dose corresponding to medium mortality.This molecule was identified to be tumor-related p63-regulated gene 1 protein.

Keywords: nickel nanoparticles; human keratinocytes; cytotoxicity; protein induction; high resolutionmass spectrometry

1. Introduction

Nickel and its compounds have become important for many industrial applications due to theirunique physical and chemical properties [1]. Nickel is widely used in modern industry for theproduction of nickel-containing alloys for coins, jewelry, internal surgical, dental devices, and stainlesssteel, or in nickel plating, as well as in the production of batteries and welding electrodes The increasinguse of nickel is directly linked with increased risk of environmental and occupational exposure [2],which leads to various health problems [3].

Nickel is one of the most potent human allergens [4], being the most common cause of contactdermatitis of the skin; about 0.5–1% of males and 5–15% of females show a positive skin reaction to patchtesting with nickel sulphate [5]. In addition, nickel is classified under Regulation (EC) No. 1272/2008as carcinogenic category 2. The facility of nickel compounds to enter the cell and alter the intracellularlevel of nickel ions is directly related with its carcinogenic potential [6]. Although nickel-inducedcarcinogenesis is known to be related to molecular events [7], the molecular mechanisms of nickelcarcinogenicity are still uncertain, and they require additional research, especially at the molecularlevel [8].

Nickel can exist in water-soluble, sulfidic, oxidic, metallic, and nanoparticulated form. Nickelcarcinogenicity is compound-dependent since only certain forms of nickel can penetrate into the cell

Nanomaterials 2020, 10, 992; doi:10.3390/nano10050992 www.mdpi.com/journal/nanomaterials

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and alter the intracellular dose [7,9]. For instance, water-insoluble nickel compounds are understoodto be much more potent carcinogens than the soluble forms [6,10]. Therefore, the identification of themolecular targets of insoluble chemical forms of nickel in human cells is critical.

Nickel nanoparticles (NiNPs) are being increasingly used in electronic applications and ascatalysts and sensors. In vitro studies in mammalian cells involving NiNPs have shown evidence of theactivation or up-regulation of cell signal pathways that are related to carcinogenicity, but the specificmechanisms are still not well understood [11]. The study of their uptake by cells as well as their toxicityassessment implies a case-by-case investigation due to the specific properties of metal nanoparticles [12].The methodologies are based on the identification of the molecular-targets of Ni-compounds and ofmolecules to be synthetized by organisms in response to Ni exposure. The analytical approaches usedto isolate, detect, and identify nickel-binding molecules were recently reviewed and discussed [13].

The goal of this work was to investigate, at the molecular level, the response of human skin cellsto the exposure to NiNPs, to correlate it with their cytotoxicity and nickel uptake, and to comparethe behavior of NiNPs with that of other chemical forms of nickel. For the purpose of the detectionand identification of the Ni-binding forms at trace levels, advanced techniques that are based onthe combination of different chromatographic techniques with the parallel elemental and moleculardetection have been developed.

2. Materials and Methods

2.1. Reagents and Chemicals

Analytical and biological reagent grade chemicals and LC-MS grade solvents were purchasedfrom Sigma–Aldrich (St. Louis, MO, USA). Nickel nanoparticles (NiNPs, ≥ 99% trace metal basis;information regarding synthetic pathway not provided), nickel sulfate (NiSO4, ReagentPlus® grade),nickel chloride (NiCl2, ReagentPlus® grade), nickel oxide (NiO, ≥99.995% Trace Metal Analysis), andnickel sulfide (Ni3S2, 99.7% trace metal basis) were used as representatives of different chemical formsof nickel throughout all of the study. Aqueous nickel solutions were prepared from a standard stocksolution of 1000 mg L−1 (Sigma–Aldrich) by dilution in ultrapure water. Ultrapure water (18.2 MΩ cm)was obtained from a Milli-Q system (Millipore, Bedford, MA, USA).

2.2. Cell Viability Assay

Adult human epidermal keratinocytes (HEKa) cells (Thermo Fisher Scientific, Waltham, MA,USA) were grown in EpiLife® Medium (Thermo Fisher Scientific) that was supplemented withcalcium in a humidified incubator with 5% CO2 at 37 C according to the manufacturer’s instructions.The cytotoxicity of nickel nanoparticles and other chemical forms of nickel to the keratinocytes cells wasquantitatively measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction assay [14], according to the International Standard Methods for BiologicalEvaluation of Medical Devices [15].

A suspension of HEKa cells was placed in a 96-well plate (200 µL and 8500 cells per well). The cellswere treated in the absence (control cells) or in the presence of increasing doses of NiNPs or Ni(II)(NiSO4, NiCl2, NiO, and Ni3S2) for 24 h. The concentration range of nickel used for each nickelcompound is shown in Supplementary Materials (Table S1), in terms of the total amount of nickeladded. Final concentrations were chosen after a first round of experiments while using a wider range.At the end of the incubation time, the medium was discarded and a new medium (200 µL) that wassupplemented with 50 µL MTT (Sigma–Aldrich) solution (5 mg mL−1 in PBS) was added to each well.Plates were incubated at 37 C for 4 h. After removing the medium, 200 µL DMSO (Sigma–Aldrich) wasadded to solubilize the formazan crystals. Finally, 25 µL of 0.1 M glycine-NaCl buffer, pH 10.5, wereadded and the plates were immediately read at 450 nm on an EON microplate automatic plate reader(BioTek, Winooski, VT, USA). Three independent experiments were carried out for every concentrationof nickel compound tested, with six replicates each.

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2.3. Preparation of Nickel Nanoparticles Suspension and Stability Test

A stock suspension of NiNPs was prepared in the medium before the incubation by accuratelyweighing a known amount of the nanopowder, followed by sonication (Branson 2510, Bransonic,Danbury, CT; nominal power and frequency: 100 W, 42 kHz ± 6%) for five minutes in order to dispersethe powder and prevent the agglomeration of the nanoparticles. Longer sonication times were notused to avoid excessive heating of the suspension. The size distribution of the NiNPs stock suspensionwas obtained by means of Single Particle Inductively Coupled Plasma Mass Spectrometry (SP-ICPMS)(Figure S1), showing a median diameter of 46 nm and no agglomerates. On the other hand, the stabilityof the NiNPs in the medium was tested. Single Particle ICPMS was utilized to obtain the nanoparticlesize distribution after 24 h of contact with the medium. No agglomerates were observed, and themedian diameter obtained (48 nm) was not significantly different than the median diameter that wasobtained for the stock suspension, confirming that the nanoparticles were stable in the medium up to24 h.

2.4. Total Nickel Determination in Cells

The HEKa cells were seeded into T75 flasks with fresh medium to final density of 2 500 cells/cm2

in 10 mL for 48 h or until the cells were 70% confluent before starting the experimental treatments.The cells were treated in the absence (control cells) or presence of 10 and 200 mg L−1 of NiNPs, 10 and50 mg L−1 of NiSO4, 1 and 50 mg L−1 of NiCl2, 50 mg L−1 of NiO, or 200 mg L−1 of Ni3S2 for 4 h and24 h. After the treatment, the growth medium was removed and the cells remaining at the bottom of theflask were rinsed and homogenized with a buffer containing sucrose 0.25 M, MgCl2 25 mM in Tris-Hcl20 mM at pH 7.4. The homogenate was re-homogenized using a Vibra-CellTM ultrasonic probe (Sonics& Materials Inc., Newtown, CT) for 30 s (at a power of 30%, repeated three times) and subjected toserial centrifugation and ultracentrifugation steps in order to obtain the different subcellular fractions(Avanti J-26 XPI, Beckman Coulter, Barcelona, Spain). In a first step, the homogenate was centrifugedat 700× g for five min. and the pellet was resuspended in buffer and kept as the “nuclear fraction”.The supernatant was recovered and ultracentrifuged at 10,000× g for 10 min. and the pellet wasresuspended in buffer and kept as the “mitochondrial fraction”. Finally, the supernatant was recoveredand centrifuged at 20,000× g for 2 h. The pellet was resuspended in buffer and kept as the “microsomalfraction”, while the supernatant was recovered and kept as the “cytosolic fraction”. This subcellularfractionation procedure has been adopted from Cox and Emili [16].

The nickel content in culture medium, cells homogenates and subcellular fractions was determinedby monitoring isotopes 58Ni and 60Ni with an Agilent 7700x ICPMS. Each sample was analysed bytriplicate. For all of the experiments carried out, analyses of control blanks (i.e., medium without NiNPsor any other form of nickel added) were performed. For the total nickel determination by ICPMS, thesignal that was obtained for 58Ni and 60Ni in the medium was at the level of the method blank.

2.5. Single Particle-ICPMS Analysis

An Agilent 7900 ICPMS (Agilent) that was fitted with platinum cones and with Single NanoparticleApplication Module for ICPMS MassHunter software (Agilent) was used for single particle analysis.The sample introduction system consisted of a concentric nebulizer and a quartz cyclonic spraychamber. 60Ni was monitored with a dwell time of 100 µs during a total acquisition time of 60 s, whilesettling time during data acquisition was eliminated. Transport efficiency was determined as 3.5% byusing gold nanoparticles (AuNPs) standard reference material with a nominal diameter of 56 nm thatwas obtained from NIST (RM 8013, Gaithersburg, MD, USA).

2.6. Size Exclusion Chromatography-ICPMS

Superdex 75 10/300 GL or Superdex 200 10/300 GL columns (GE Healthcare, Pittsburgh, PA,USA) were coupled to an Agilent 7700x ICPMS (Agilent) instrument that was fitted with platinum

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cones. Chromatographic separations were performed by using a model 1200 series HPLC pump(Agilent) as a delivery system. The exit of the column was connected in series to an UV-visible detector(Agilent) and to the nebulizer of the ICPMS instrument that was equipped with a collision/reactioncell. Table S2 lists the operational conditions (Supplementary Materials). The Superdex 200 columnwas calibrated in order to assign a molecular weight to each peak obtained in the samples. Proteinstandards (thyroglobulin, ferritin, transferrin, superoxide dismutase, myoglobin, metallothionein, andcobalamin, purchased from Sigma–Aldrich) of known molecular weight were injected onto the sizeexclusion column under the same conditions as the samples. The obtained linear relationship betweenthe logarithm of the molecular weight and the retention time was used for column calibration.

2.7. Hydrophilic Interaction Liquid Chromatography-ICPMS

Fractions that were collected from Size Exclusion Chromatography (SEC) column were pooled,concentrated by freeze-drying (freeze dryer Crios, Cryotec, Saint-Gély-du-Fesc, France), resuspendedin ammonium acetate 100 mM pH 7.4, and injected into a SeQuant® ZIC®-cHILIC HPLC column(3 µm, 100 Å) (Millipore) coupled to an Agilent 7900 ICPMS (Agilent) fitted with platinum cones.A 1260 Infinity Bio-Inert HPLC pump (Agilent) was used as the delivery system. The exit of the columnwas connected to the nebulizer of the ICPMS instrument that was equipped with a collision/reactioncell. Table S3 lists separation program and operational conditions (Supplementary Materials).

2.8. Hydrophilic Interaction Liquid Chromatography-ESI-FT-MS/MS

The same Hydrophilic Interaction Liquid Chromatography (HILIC) column was directly connectedto an Ultimate 3000 RSLC system (Thermo Fisher Scientific, Germering, Germany) that was coupledto an Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA, USA) high resolution massspectrometer operated in positive mode. The latter was fitted with an Advion Triversa NanoMateion source, operated in LC coupling and fraction collection into wells mode. Basically, the TriversaNanoMate was used to partly split the LC flow to a chip-based nanoelectrospray, while most of theflow was directed to a 96-well plate for fractions collection. Post column acidification was achieved bythe addition of a 30% formic acid methanol solution (v/v) to the LC flow at a rate of 10 µL min−1 bythe mean of a syringe pump and a tee connector followed by a mixing loop (250 mm of a 250 µm IDpeek tubing). This post column acidification was applied from 25 to 45 min. over the chromatogram.At this stage, the MS acquisition method consisted in a full scan detection using a resolution of 120,000.Fractions containing a Ni compound were collected and analysed by nanoESI-MS. A full scan massspectrum was first acquired at a resolution of 120,000 and then MS/MS scans were performed forthe multiply charged precursor ion while using HCD fragmentation at different normalized collisionenergy (NCE) ranging from 30 to 45%. The MS/MS scan that served for fragment identification wasobtained at a NCE of 40%.

2.9. Top-Down Protein Sequence Identification

The acquired MS/MS spectrum was first converted from RAW to MzXML format using thesoftware MSconvert (ProteoWizard Software Foundation). It was then deconvoluted using MS-Deconv(available online: http://bix.ucsd.edu/projects/msdeconv/) and the protein identification was thenachieved while using the Top-Down proteomic software MS-Align+ (available at: http://bix.ucsd.edu/projects/msalign/msalign_manual.html) [17] and a human FASTA file obtained from Uniprot(https://www.uniprot.org). Finally, the candidate protein that was identified through the untargetedapproach was evaluated through a targeted approach using the software ProSight Lite (ProteomicsCenter of Excellence, Northwestern University, IL, USA).

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3. Results and Discussion

3.1. Evaluation of the Cytotoxicity of Nickel Nanoparticles and Other Nickel Compounds Towards HumanSkin Cells

The viability of human skin cells exposed to NiNPs was evaluated and compared with that afterexposure to other chemical forms of nickel (NiSO4, NiCl2, NiO, and Ni3S2). For this, model humanskin cells were incubated with the different chemical compounds in a range of concentrations and thecytotoxicity was quantitatively measured by an MTT assay, as described in the Experimental section.Adult human epidermal keratinocytes were chosen as the cell line model. Keratinocytes are wellcharacterized cells and they were used as cell allergy models elsewhere [18]. After the MTT assays,the dose-dependent cellular toxicity was determined for each nickel compound. Figure 1 shows theobtained dose-dependent curves.

Figure 1. Dose-response curves obtained for the nickel compounds tested: (a) NiNPs; (b) NiSO4;(c) NiCl2; (d) NiO; and, (e) Ni3S2. The curves are compared in (f).

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For NiSO4, NiCl2, NiO, and Ni3S2, the dose-response curves could be fitted to sigmoid curvesallowing for the determination of the LD50 values (Table 1). Among the compounds tested, NiOpresented the highest toxicity, followed by the two soluble compounds (NiSO4 and NiCl2), whichshowed similar LD50 values. Surprisingly, the lowest toxicity was observed for nickel subsulfide.Indeed, in an experiment testing 11 Ni compounds, Ni3S2 was found to be more toxic towards AS52cells than NiSO4, NiCl2, and NiO [19]. In any case, the results showed that even the least toxic Ni3S2

show certain toxicity towards human skin cells.

Table 1. LD50 values obtained for the studied nickel compounds.

Compound NiNPs NiSO4 NiCl2 NiO Ni3S2

LD50, mg L−1 - 56.4 58.3 49.2 249.5

For NiNPs, the shape of the dose-response curve was different. The curve was linear, meaning thatNiNPs exert a specific toxic effect towards human keratinocytes and that even low doses represent arisk. This kind of behavior, where a threshold dose is not observed, is believed to be typical of genotoxiccarcinogens [20,21], although some controversy remains [22,23]. In our study, the cytotoxicity resultsagreed with those that were obtained for human skin cells (A431) exposed to nickel nanoparticles [24].The genotoxic potential of metallic NiNPs was reported for human skin cells (A431) [24] and theircarcinogenicity for mouse epidermis cells (JB6 cells) [25]. Note that the NiNPs used in the study withmouse epidermis cells are significantly bigger (92.3 nm) than those used in study with human skincells and in the present one (52 and 46 nm, respectively). On the other hand, some oxide nanoparticleswere reported to show a similar cytotoxicity behavior: TiO2 NPs in mouse fibroblast cells (L929) [26],silicon dioxide (SiO2) nanoparticles in human monocytes (THP-1) [27], and NiO NPs in humanbronchoalveolar carcinoma-derived cells (A549) [28]. In any case, in the present study a cell mortalityof around 50% was observed when the incubation was performed with a nickel concentration of200 mg L−1.

3.2. Determination of the Nickel Uptake

Human epidermal keratinocytes were incubated with NiNPs and the two soluble nickel salts(NiCl2 and NiSO4) for 24 h at a nickel dose corresponding to a medium cell mortality in order tostudy the nickel cellular uptake, as observed in the cytotoxicity study, i.e., 200 mg L−1 for NiNPsand 50 mg L−1 for the two nickel salts. After exposure, the medium was recovered, the cells werewashed out with a buffer, and the total nickel content in both fractions was determined by ICPMS.For the experiments with both nickel salts, around 0.2% of the nickel mass added was found in thecells, whereas the rest of the nickel remained in the medium (Table 2). Good recoveries (>95%) wereobtained for both of the experiments.

Table 2. Total amount of nickel found in the medium and in cells treated with NiNPs and soluble nickelcompounds at 24 h with a nickel dose corresponding to medium mortality.

Compound Ni MassAdded, µg

Ni Mass inMedium, µg

Ni Mass inCells, µg (%) Rec, % Ni Mass in Cytosol,

µg (%)

NiNPs 1000 42.6 ± 0.1 37.1 ± 0.2(3.71 ± 0.02) 8 ± 1 1.96 ± 0.05

(0.20 ± 0.01) *

NiCl2 250 248 ± 14 0.59 ± 0.01(0.24 ± 0.01) 99 ± 5 0.53 ± 0.01

(0.21 ± 0.01) *

NiSO4 250 240 ± 2 0.54 ± 0.01(0.21 ± 0.01) 96 ± 1 0.42 ± 0.01

(0.17 ± 0.01)

* Values with no significant differences among groups with a level of confidence of 95%.

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Regarding the experiment with NiNPs, the corresponding amount of nickel found in the cellswas 20 times higher. According to the obtained results, the cells took up 3.71% of the nickel addedto the culture. However, the recovery obtained (Ni mass found in the medium plus Ni mass foundin cells) for this experiment was very low: 8%. These results can be explained by the fact that thenanoparticles are in suspension and they may settle at the bottom of the flask during cultivationexperiments. Therefore, after the treatment, they are not removed when the medium is recovered, butwhen the cells are rinsed and homogenized. An experiment in which NiNPs were incubated with amedium under the same conditions but without the presence of cells was performed in order to verifythis hypothesis. It was found that 91% of nickel nanoparticles stuck to the bottom of the flask andthe amount of nanoparticles recovered with the buffer was similar to that obtained in the experimentwith cells. As this fact might bias the interpretation of the results obtained for the experiment withNiNPs, it was necessary to fractionate the cell and study the distribution of the nickel content amongthe different subcellular fractions in order to confirm that the nickel from NiNPs was taken up by thecells and, at the same time, to identify the target organelles of nickel.

Keratinocytes were incubated with NiCl2 at a dose corresponding to a medium mortality during24 h. After the treatment, the homogenate of cells was removed from the flask and the followingsubcellular fractions were collected after serial centrifugation and ultracentrifugation: nuclear fraction,mitochondrial fraction, microsomal fraction, and cytosolic fraction. ICPMS determined the total nickelcontent in each fraction. It was found that nickel was mainly located inside the cytosol (almost 90%of the total nickel present in the initial homogenate), whereas the amount of nickel in the nucleuscorresponding to 5% of the total Ni is so small that the assessment of its chemical form is below thecapacity of any state-of-the art analytical technique, including this developed in this study. This resultis in good agreement with other studies that were conducted with HaCaT keratinocytes that showedthat nickel (in the form of NiCl2) was mainly accumulated in the cytosolic fraction [29,30]. RegardingNiNPs, the nickel present in the nucleus and in the mitochondria account for 10% of the intracellularnickel and the nickel amount between nucleus and mitochondria was not discriminated. The cytosolwas found to be the target organelle of nickel and it was the objective of the subsequent study, accordingto these results.

A significant amount of nickel (1.96 µg) was found in the cytosol of cells that were treated withNiNPs, confirming that keratinocytes were able to take up nickel when put in contact with a suspensionof NiNPs and store it in the cytosol. In comparison with the results obtained for the cytosol of cellstreated with soluble nickel salts (0.21 and 0.17 µg for NiCl2 and NiSO4, respectively), the amount ofnickel found was four times higher, which correlates with the amount of nickel added: 1000 µg for theexperiment with NiNPs vs. 250 µg for the experiments with nickel salts. However, the mass of nickelthat was found in cytosols was significantly lower than the mass of nickel that was determined in thenickel homogenate (37.1 µg, Table 2), which again suggests that the majority of nickel found in thehomogenate does not correspond to intracellular nickel.

In the next step, ICPMS determined the total nickel content in cytosols from cells treated withNiNPs and the other nickel compounds at two different doses (low and medium mortality) and twodifferent incubation times (24 h and 4 h) after ultracentrifugation. For comparison purposes, resultsobtained results were normalized and expressed as % of nickel found in cytosol with respect to thetotal amount of nickel added (Figure 2). As expected, the percentage of nickel present in cytosols ofcells treated with NiNPs increased when increasing the incubation time from 4 h to 24 h, a behaviorthat was also observed for the other nickel compounds. However, for the same incubation time, ahigher percentage of nickel was found in cytosols of cells treated with the lower dose of nickel: 0.23%of nickel for a dose corresponding to low mortality against 0.20% of nickel for a dose corresponding tomedium mortality. This behavior, which was not observed for the soluble nickel compounds, NiCl2and NiSO4, suggests that the toxicity of NiNPs is not related to the amount of intracellular Ni.

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Figure 2. The fraction of nickel (with respect to the total nickel added to the culture) found in thecytosol of cells treated with different nickel compounds at different doses and incubation times. * Valueswith no significant differences among groups with a level of confidence of 95%.

The results that were obtained for NiO and Ni3S2 (Table S4) show that the highest amount ofnickel in cytosols was found after the treatment with NiO, which agrees with the fact that NiO was thecompound that showed the highest toxicity among all of the studied compounds (Table 1). However,no significant differences were found between the percentages of nickel found in cytosols that weretreated with Ni3S2 and NiSO4, despite their different toxicities, which makes us conclude that thetoxicity of the different nickel compounds is not solely explained by the amount of nickel presentin cytosol.

3.3. Physicochemical Form of Ni in Cell Cytosols

Even though the results that were obtained in the experiments with NiNPs clearly confirmedthat the cells took up a significant amount of nickel, the physicochemical form of this nickel presentin the cytosol is unclear. From a toxicological and molecular point of view, it is essential to knowwhether nickel is able to enter the cell in the form of nanoparticles. The cytosols treated with NiNPswere analysed directly by single particle inductively coupled plasma mass spectrometry (SP-ICPMS)in order to answer this question. This technique can discriminate between the nanoparticulate andthe dissolved form [31,32]. The time scan obtained only showed a steady signal characteristic ofthe presence of Ni in dissolved form [33] (Figure S2). However, pulses with intensities above thebackground, typical of the presence of nanoparticles, were not observed. If NiNPs were presentin the cell, then the use of SP-ICPMS after sonication would still allow the detection of individualnanoparticles without agglomeration, as it was recently shown in literature [34,35].

Consequently, it can be concluded that all of the nickel present in cytosols comes from theoxidation/dissolution of the nanoparticles. Other metallic nanoparticles were also reported to dissolvein cellular growth media, such as silver nanoparticles (AgNPs) in Dulbecco’s Modified Eagle Medium(DMEM) [36–38] or in Rosewell Park Memorial Institute (RPMI) media [39]. The fact that all nickelpresent in cytosols was found in its dissolved form due to the dissolution of NiNPs perhaps explainingthe results observed in this study. For the nickel soluble salts, all of the nickel is available for cellsimmediately upon addition (and hence the higher the concentration, the higher nickel available),whereas, in the case of NiNPs, the availability of nickel ions depends on the dissolution of nanoparticles.Therefore, the uptake of nickel by cells is ruled by a kinetic process and, as it was shown for othermetallic nanoparticles, such as AgNPs, the ion release rate increases when the concentration of

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nanoparticles decreases [40]. The relative number of nickel ions released and, hence, available for thecells, was higher in the case of the experiment with a lower concentration of NiNPs (corresponding tolow mortality) when compared with the experiment with a higher concentration, which explains whythe percentage of nickel found was higher in the former experiment (Figure 2). On the other hand,the cytotoxicity of NiNPs might be linked to the dissolution and release of metallic ions, as it wassuggested for other metallic nanoparticles, such as AgNPs [41], which may also explain the observedlinear dose-response curve (Figure 1). For instance, a similar behaviour that was found for ZnO NPswas related to the cytosolic concentration of Zn2+ as well as to an apoptotic death pathway [42]. At thisstage of research, the cause of cell death remains unknown, although apoptosis was shown to be theunderlying mechanism used by ZnO NPs to induce cell death.

3.4. Separation of Ni-Binding Compounds

Once in the cytosol, nickel is likely to be bound to molecules already present or produced bythe cells. The characterization and identification of these molecules is necessary to understand themolecular mechanisms of nickel toxicity. In this context, different analytical approaches were developedin order to separate, characterize, and identify the Ni-binding compounds present in cytosols of cellstreated with NiNPs and the other nickel compounds.

In the first approach, size exclusion chromatography was used for the separation of the proteins.Cytosols that were treated with NiNPs, NiCl2, and NiSO4 under the same conditions that were used inthe study of the nickel uptake were injected onto a size exclusion column Superdex 200 coupled toUV-Vis and ICPMS detectors. Since similar chromatograms were obtained for the two nickel (NiCl2and NiSO4) salts, the chromatograms obtained for NiNPs were only compared with NiCl2. Figure 3shows the chromatograms obtained of cytosols of cells treated with both nickel compounds at differentconcentrations and incubations times. Regarding the statistical significance, the reproducibility of SECchromatograms is +/−5% in terms of intensity and 2% in terms of elution time. The chromatograms(peaks) fitting this range were considered to be identical and are marked with an asterisk in Figure 3.

All of the chromatograms show a peak at a retention time of around 30 min. (peak II, correspondingto a compound of 1.4 kDa), whereas an additional peak eluting at around 26 min. (peak I, correspondingto a compound of 6.8 kDa) was only observed under some conditions. When comparing cytosols thatwere treated with both compounds, chromatograms were similar, except for the case of cells that weretreated for 4 h at a dose corresponding to medium mortality, where the intensity was significantlyhigher for NiNPs (Figure 3a). This is in good agreement with the amount of Ni found in cytosols forthe experiment with NiNPs (1.60 µg) as compared with the one that was found for the experimentwith NiCl2 (0.09 µg). On the other hand, the differences between nickel doses were significant, as canbe observed in Figure 3b. Peak I was observed for cytosols of cells that were treated with a nickel dosecorresponding to medium mortality, whereas it was not observed, or it was not significant in cytosolsof cells treated with a low nickel concentration (corresponding to low mortality), suggesting that thiscytosolic compound is involved in the mechanisms of nickel toxicity. In addition, in the case of NiNPs,a significant increase in the intensity of peak II was observed for an increased nickel dose (1.96 and0.12 µg of Ni found in cytosols for medium and low toxicity experiments, respectively), which was notthe case in the case of cytosols that were treated with NiCl2. Finally, the comparison of chromatogramsshowed that the intensity of peaks I and II increased as a function of incubation time. This is especiallysignificant in the case of peak I, whose intensity is higher than that of peak II for cytosols treated at adose of medium mortality during 24 h (0.53 and 1.96 µg of Ni found in cytosols for NiCl2 and NiNPsexperiments, respectively).

In addition, cytosols from control cells (i.e., incubated under the same conditions, but withoutthe presence of nickel) were spiked with Ni2+ and the cytosolic compounds were separated by sizeexclusion chromatography in the same conditions as the samples. Taking a look to the chromatogramobtained (Figure 4), peak I, which is present in the sample of cells that were treated with NiNPs at anickel dose corresponding to medium mortality and 24 h incubation time was not observed in the

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cytosol of control cells spiked with Ni2+. This result shows that the compound that is responsible forthe presence of this peak is not naturally present in cytosol, and it is only expressed by cells understress in the presence of high doses of nickel. The peak (black line) observed in Figure 4 correspondsto a nickel-binding species that is present in the control cytosol of keratinocytes. For this reason, itsidentity was not investigated.

Figure 3. Chromatograms obtained by SEC-ICPMS for cytosols of cells treated with NiNPs and NiCl2at two different nickel doses (corresponding to medium and low mortality) and two incubation times(24 and 4 h). (a) Comparison between treatments with NiNPs and NiCl2; (b) comparison betweentreatments at medium and low mortality; and, (c) comparison between treatments at 24 and 4 h.Asterisks indicate a pair of chromatograms (or relevant peaks) without statistical differences betweenthem. The chromatograms were normalized for the purpose of comparison assuming the intensity ofthe highest peak in either chromatogram as 100%.

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Figure 4. Chromatograms obtained by SEC-ICPMS for cytosols of cells treated with NiNPs at a mediummortality dose for 24 h (red line) and cytosols of untreated cells spiked with Ni2+ (black line).

3.5. Identification of the Ni-Binding Compound Expressed by Keratinocytes

An analytical strategy was developed in order to identify the compound that was differentiallyexpressed by human keratinocytes in the presence of NiNPs. The developed strategy consisted ofa second chromatographic separation step coupled in parallel with ICPMS and a high-resolutionelectrospray mass spectrometer (ESI-FT-MSn). ICPMS allowed for monitoring the nickel signal of thecomplex, whereas ESI-FT-MS/MS provided the identity of the nickel-binding protein.

A better resolution of the two peaks observed is needed before performing a second dimensionchromatographic step for the identification of the nickel-binding compounds corresponding to peak I.It was achieved by using a Superdex 75 column. At the same conditions of carrier composition andflow rate, a chromatogram with the peaks well resolved was obtained for cytosols of cells treated withNiNPs at a dose of medium toxicity and 24 h incubation time (Insert in Figure 5). In addition, cytosolsfrom cells that were treated with NiO and Ni3S2 were also analyzed by SEC-ICPMS with the Superdex75 column (data not shown). In the case of NiO, the presence of a cytosolic compound differentiallyexpressed by the cells was observed, even after 4 h of incubation time, which was not the case of theexperiments that were carried out with the other nickel compounds. The fact that NiO was found to bethe nickel compound with the highest cytotoxicity might explain the expression of this protein by thecells, even at low incubation time. On the other hand, the peak I was not observed for the treatmentswith Ni3S2, even at a dose of medium mortality and 24 h of incubation time, which might be relatedwith the low toxicity that was found for this compound.

Figure 5. Chromatogram obtained by HILIC-ICPMS for SEC (Superdex 75, 58Ni detection) fraction(shown in the inset) for a cytosol of cells treated with NiNPs for 24 h at a nickel dose corresponding tomedium mortality.

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For the identification of the Ni-binding compound expressed by keratinocytes, the cytosol of asample that was treated with NiNPs at 24 h incubation time at a dose corresponding to medium cellviability was fractionated on the Superdex 75 SEC column. The fraction corresponding to peak I wascollected, concentrated, and subsequently analyzed by HILIC coupled to ICPMS. HILIC provides anefficient separation for small polar compounds, keeping the metal-biomolecule complex intact [43].Figure 5 shows the chromatogram obtained for the SEC fraction collected from cytosols of cells treatedwith NiNPs; the main peak corresponds to the nickel-binding compounds of interest.

An aliquot of this SEC fraction was analyzed by HILIC coupled to ESI-FTMS under the sameseparation conditions. Unfortunately, no signal of a Ni-bioligand compound was obtained, whichwas probably due to the low protein concentration in the sample. However, the use of in chip-basedelectrospray ionization (NanoMate) allowed for the splitting of the flow at the exit of the column,the collection of the fraction at the retention time of the Ni-compound, and its subsequent analysisin chip-based infusion MS. Figure 6 shows the deconvoluted mass spectrum corresponding to apolypeptide of a molecular weight of 5810.13344 Da. The comparison of the molecular weightmeasured vs. the theoretical one (5810.0996 Da) resulted in a mass difference of 5.82 ppm. Notethat a post column acidification was applied in order to remove Ni from the complex to facilitatethe electrospray ionization at the retention time of the Ni-compound, as discussed elsewhere for theidentification of metal-binding proteins by ESI MS [44]. Consequently, the observed molecular masscorresponds to the polypeptide ligand. Figure 6b shows the MS/MS fragmentation spectrum that wasobtained by using high-energy collisional dissociation (HCD) fragmentation mode.

Figure 6. Cont.

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Figure 6. (a) Deconvoluted mass spectrum obtained for the SEC-HILIC Ni-containing fraction bynano-electrospray mass spectrometry (ESI-FT-MS) (infusion). The cluster of peaks at m/z of 1453.2888Da corresponding to a quadruple charged molecule is shown as insert (b) MS2 fragmentation spectrumobtained by high-energy collisional dissociation (HCD) for the m/z 1453. (c) zooms on the 16 peak ionshaving served to the identification of the sequence.

The MS and MS2 fragmentation data (the whole list of fragments obtained is available asSupplementary File) were processed through an untargeted Top-Down proteomics approach anddifferent fragments were obtained (Table 3). The MS2 was zoomed at the vicinity of each of the fragmentsshown in Table 3 and, thus, 16 zooms were collected and are shown in Figure 6c. These fragmentsallowed for the identification of the polypeptide sequence that is shown in Figure 7. The sequenceis related to a protein expressed by human epidermal cells: tumor protein p63-regulated gene 1(TPRG-1) [45]. An open question is whether this polypeptide corresponds to the truncated isoform ofthe TPRG1 protein or is an enzymatic cleavage fragment. Anyhow, this question is secondary and itshould not eclipse the finding that a TPRG1-related polypeptide is expressed in response to the NiNPsstress. Moreover, the truncated isoform (or its enzymatic cleavage fragment) binds strongly to nickelto let the complex pass through an HPLC column. To our knowledge, such stable polypeptide-metal

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complexes have not been reported from products of enzymatic cleavage, so the occurrence of a newtruncated isoform of the TPRG1 protein has been assumed. Note that the binding of Ni occurs in theabsence of a H2C2, which is a common Zn-finger domain that also binds Ni(II). The key to this bindingis probably the presence of histidine residue and the fact that there is only one make the binding weak.Note that the binding of Ni to polypeptides containing a single histidine was reported elsewhere [46].

Table 3. MS2 data having allowed the identification of the Ni-binding polypeptide.

Ion Type Theoretical Mass Observed Mass Mass Difference (Da) Mass Difference (ppm)

B4 426.2227 426.2264 0.00368 8.6B8 876.4566 876.4566 −0.00002 −0.023B9 963.4886 963.4879 −0.00069 −0.72

B12 1208.5898 1208.5947 0.00493 4.1B14 1421.7124 1421.7138 0.00147 1.0B15 1478.7338 1478.7388 0.00499 3.4B16 1606.8288 1606.8423 0.01354 8.4B19 1979.0483 1979.0335 −0.01484 −7.5B23 2421.2910 2421.2671 −0.02396 −9.9B45 4902.6000 4902.5637 −0.03635 −7.4Y10 1069.5556 1069.5532 −0.00239 −2.2Y11 1126.5771 1126.5867 0.00964 8.6Y20 2156.1490 2156.1305 −0.01852 −8.6Y33 3617.8915 3617.8719 −0.01961 −5.4Y34 3730.9756 3730.9461 −0.02947 −7.9Y47 5046.6786 5046.6357 −0.04290 −8.5

Figure 7. Amino acids sequence coverage of the induced nickel-binding polypeptide.

4. Conclusions

This study demonstrates, for the first time, that the dose-response curve obtained in the cytotoxicityassays for the exposure of human skin cells (keratinocytes) to NiNPs shows a linear behavior that istypical of genotoxic carcinogens, and it is different from the response to other Ni species. From thechemical point of view, at a dose corresponding to medium mortality, the exposure to NiNPs leads tothe release and accumulation of Ni2+ ions in the cytosol and the biosynthesis of a 6 kDa nickel-bindingmolecule related to the p63-regulated gene 1 protein. It should be noted that the exposure of humancells to genotoxic carcinogen usually leads to the expression of various metabolites, but this studyonly focused on molecular targets of nickel. The improvement of instrumental technology providinglower detection limits will unavoidably lead to the detection of new proteins that are involved inmolecular mechanisms of nickel toxicity. At the same time, future research should be oriented towardscomparative proteomics or metabolomics in order to investigate the induction/suppression of ligandsthat are not chemically linked to Ni.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/5/992/s1,Supplementary File 1: Figure S1: Nanoparticle size distribution obtained by single particle-ICPMS for the stocksuspension of NiNPs, Figure S2: Time scan obtained by single particle-ICPMS for cell cytosols treated withNiNPs, Table S1: Concentration range of nickel used for each nickel compound during the cytotoxicity assays,Table S2: Operational conditions of SEC-ICPMS analysis, Table S3: Operational conditions of HILIC-ICPMSanalysis, Table S4: Total amount of nickel found in the medium and in cytosols treated with NiO Ni3S2 at 24 h

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with a nickel dose corresponding to medium mortality. Supplementary File 2: List of fragments obtained fromMS2 fragmentation.

Author Contributions: Conceptualization, J.J.-L., J.S. and R.Ł.; Funding acquisition, J.J.-L. and J.S.; Investigation,J.J.-L., S.G. and G.A.; Methodology, J.J.-L., S.G., G.A., C.B. and J.S.; Supervision, J.S. and R.Ł.; Writing—Originaldraft, J.J.-L.; Writing—Review & editing, J.J.-L., S.G., G.A., C.B., J.S. and R.Ł. All authors have read and agreed tothe published version of the manuscript.

Funding: This work has received funding from the European Union’s Horizon 2020 research and innovationprogram under the Marie Sklodowska-Curie grant agreement no. 660590.

Acknowledgments: The authors gratefully acknowledge Niurka Dariela Llópiz for her excellent technicalassistance. The funding of the FT MS platform by the EQUIPEX ANR -11-EQPX-0027 MARSS projectis acknowledged.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of thestudy; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision topublish the results.

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